Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/26—Preparation of nitrogen-containing carbohydrates
  • C12P19/28—N-glycosides
  • C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/803—Physical recovery methods, e.g. chromatography, grinding
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/8215—Microorganisms
  • Y10S435/822—Microorganisms using bacteria or actinomycetales

Definitions

• This invention relates to a process for preparing vitamin B (cyanocobalamin) and more particularly to an improved process for converting the LLD active components in a fermentation broth to vitamin B
• vitamin B cyanocobalamin
• Prior to this invention numerous methods were known for preparing and recovering vitamin B Unfortunately these previously known processes suffered the disadvantages of producing recoverable vitamin B in relatively low yield and/ or high impurity. By the process of this invention, however, vitamin B is obtained in both high yield and purity.
• the process of this invention comprises culluring a vitamin B producing strain of microorganism in a cobalt containing nutrient medium deficient in cyanide ions, extracting the LLD active components from the medium, treating the extract with a particulate cation exchanger, whereby the LLD active components are ad- 'sorbed on the exchanger, eluting said components from the exchanger, and treating the eluate simultaneously with a source of cyanide ions and light to convert the LLD active components to vitamin B (By LLD active components are meant substances which give a positive result when tested by the assay procedure described in US. Patent No. 2,703,302.)
• microorganism which produces vitamin B (when cultured in a nutrient medium containing cobalt and cyanide ions) can be used in the process of this invention.
• Such microorganism include Streptomyces, such as Streptomyces griseus, Streptomyces aureofaciens, Streptomyces albidoflavus, Streptomyces antibioticus, Streptomyces colombiensis, Streptomyces fradiae, Streptomyces roseochromogcnus and Slreptomyces olivaceus; Aerobacter, such as Ael'obacter aerogenes; Ashbya, such as Ashbya gossypii; Mycobacterium, such as Mycobacterium phlei, Mycobacterium smegmatis and M ycobaclerium tuberculosis; and preferably Propionibacterium, such as Propionibacterium freudcnreichii.
• sources of assimilable carbon there is employed the usual sources of assimilable carbon, nitrogen and cobalt.
• sources of assimilable carbon there may be used: (1) carbohydrates, such as glucose, fructose, sucrose, maltose, dextrins and soluble starches; (2) substances containing carbohydrates, such as corn steep liquor and grain mashes; (3) polyhydric alcohols, such as glycerol; (4) fats, such as lard oil, soybean oil, linseed oil, cottonseed oil, peanut oil, coconut oil, corn oil, castor oil, sesame oil, palm oil, mutton tallow; sperm oil, olive oil, tristearin, triolein and tripalmitin; and (5) long chain fatty acids such as stearic acid, palmitic acid, oleic acid, linoleic acid and myristic acid.
• Sources of available nitrogen include: (1) organic nitrogen compounds, such as proteinaceous materials, e.g., soybean meal, fish meal, casein, whey or whey concentrates, yeast, amino acids and liver cake; and (2) inorganic compounds, such as nitrates or ammonium compounds.
• organic nitrogen compounds such as proteinaceous materials, e.g., soybean meal, fish meal, casein, whey or whey concentrates, yeast, amino acids and liver cake
• inorganic compounds such as nitrates or ammonium compounds.
• Assimilable cobalt may be supplied to the nutrient maximrn in a variety of forms. It may be provided in the form of cobalt salts, such as cobalt chloride or cobalt nitrate; or it may be provided in the form of organicallybound cobalt, such as a cobalt-containing yeast, preferably one containing a relatively high proportion of cobalt (i.e., about or more parts per million); or it may be naturally present in one of the sources of carbon or nitrogen, such as beet molasses.
• cobalt salts such as cobalt chloride or cobalt nitrate
• organicallybound cobalt such as a cobalt-containing yeast, preferably one containing a relatively high proportion of cobalt (i.e., about or more parts per million); or it may be naturally present in one of the sources of carbon or nitrogen, such as beet molasses.
• the nutrient medium may, of course, contain any of the additional components usually found in such solutions (except a source of cyanide ions).
• additional components e.g., antifoam agents (e.g., lard oil and octadecanol), metallic cations, such as potassium, calcium, magnesium and iron (which may be present in the crude materials used in the nutrient medium), and phosphates (which may be added as inorganic phosphate).
• the fermentation process may be carried out at any normal temperature, such as one from about 20 C. to about 40 C.
• a source of oxygen or air should also be present. This aeration can be accomplished by bubbling air (or oxygen) through the medium during the fermentation period or by agitating the medium, thereby exposing a large surface thereof to the atmosphere. If the microorganism is anaerobic in nature, the aeration step is omitted. However. in some instances an aeration step towards the end or after the fermentation period has proven to be beneficial.
• the LLD active components in the fermentation broth are recovered.
• This recovery may be accomplished in a number of ways known to the art. Preferably this recovery is achieved by separating the cells by centrifugation or filtration and then rupturing the cells by heat treatment, acid treatment, and/or slurrying in 50% aqueous acetone.
• the thus released LLD active components are separated from the ruptured cells by extraction into an aqueous medium.
• steam may be added to the suspension of the separated cells to raise the temperature to about 70 C.
• the aqueous extract contains the LLD active components, a portion of which is in the form of hydroxocobalamin.
• some of the LLD activity is present in forms other than hydroxocobalamin and it is in the conversion of these active by-products to vitamin B that the process of this invention is advantageous over any previously known process in the art.
• the extract is then acidified, if necessary, to a pH in the range of about 2 to about 6 (preferably about 2 to about 3) by treatment with an acid, such as a mineral acid as exemplified by hydrochloric and sulfuric acid and the acidified extract is then treated with a particulate cation exchanger, preferably at a temperature of about 20 C. to about 40 C.
• an acid such as a mineral acid as exemplified by hydrochloric and sulfuric acid
• a particulate cation exchanger preferably at a temperature of about 20 C. to about 40 C.
• Suitable cation exchangers for use in the process of this invention include those cation exchangers, especially cation exchange resins, particularly of the carboxylic acid type, priorly used for the adsorption of streptomycin and other basic antibiotics.
• cation exchangers especially cation exchange resins, particularly of the carboxylic acid type, priorly used for the adsorption of streptomycin and other basic antibiotics.
• cation exchangers particularly of the carboxylic acid type, priorly used for the adsorption of streptomycin and other basic antibiotics.
• methacrylate carboxylic resins such as those sold under the trademarks: Amberlite IRC-SO and Amberlite XE-89.
• the treatment of the extract with the cation exchanger may be carried out either batchwise or continuously, using one or more cation exchange columns, tanks or other vessels. Cation exchange columns are preferred, however, because of their efiiciency, ease of operation and compactness.
• the extract is passed, preferably downflow, through the cation exchanger, in its hydrogen form, until substantially all of the LLD active components are adsorbed on the exchanger.
• One column may be used or a plurality of columns connected in series can be employed to assure complete adsorption of the active material.
• the active material is then eluted from the exchanger by treatment with a basic material.
• a basic material Preferably an inorganic basic material is used, such as an aqueous solution of an alkali metal hydroxide (e.g., sodium hydroxide) or ammonia.
• alkali metal hydroxide e.g., sodium hydroxide
• ammonia particularly preferred, however, are salts of strong bases and weak acids, such as an alkali metal carbonate (e.g., sodium carbonate and sodium bicarbonate) since these reagents give a low or a negative heat of reaction thereby minimizing the chance of destruction of the active materials during elution from the resin.
• the elution may be carried out continuously in the columns themselves, if such are used, or batchwise by dumping the contents of the columns in tanks and adding the basic material to the tanks.
• Elution is continued until the eluate is at a basic pH, preferably about 7 to about 10, thereby assuring substantially complete removal of the active materials from the exchanger. If desired, the exchanger can then be washed with water and the Wash combined with the eluate.
• the eluate is then treated with light and a source of cyanide ions.
• the term light means electromagnetic waves of wavelength of about 3600 to about 8000 Angstroms.
• the illumination may be supplied by use of white fluorescent bulbs.
• any source of cyanide ions may be used and thus h drogen cyanide itself is not excluded, the preferred sources of cyanide are the alkali metal salts, such as potassium cyanide. Since this cyanide treatment is conducted under basic conditions the hazard due to hydrogen cyanide fumes is thereby minimized.
• the cyanide may be added before the irradiation step or may be, and preferably is, added intermittently during said step.
• the irradiation and cyaniding step takes from about 2 to about 30 hours and results not only in the conversion of the hydroxocobalamin to vitamin B but also in the conversion of various LLD active by-products to the desired vitamin B
• the resulting vitamin B containing solution is then treated in the usual way to recover the vitamin B therein.
• One such method entails the extraction of the vitamin B into an organic solvent (e.g., a phenol-benzene solvent) in the manner known to those skilled in the art.
• EXAMPLE 1 F ermentation.A sterile aqueous medium consisting of 7.6% beet molasses, 1.5% yeast autolysate solids, 1.0% corn steep liquor, and 2.0% CaCO contained in a stainless steel fermentation vessel, is inoculated with an active culture of Propionibacterium freudenreichii. Following a four day fermentation period at 30 (pH controlled at pH 7.0-8.0 with sodium hydroxide), the bacterial cells are harvested by centrifugation.
• Ads0rpti0n.Two liters of concentrate obtained in step b are acidified to pH 2.6 by addition of H 50 and filtered to remove insoluble matter.
• a chromatographic column, 600 x 40 mm., is packed with wet Amberlite IRC-SO resin in its hydrogen form to a height of 22 inches. Filtered concentrate is passed downward through the column at a rate of 25 ml./min., followed by a rinse with tap water acidified to pH 2 with sulfuric acid. Both effiuent concentrate and wash are virtually free of LLD active matter.
• EXAMPLE 2 Fermentation-An active culture of P. freudenreichii is used to inoculate a heat sterilized aqueous medium containing 2% yeast autolysate solids, 1.0% corn steep liquor, 7.6% beet molasses, 1.0% Staleys Sauce No. 3 (a soy sauce), 5 p.p.m. cobalt (as cobalt sulfate) and 2% CaCO A pH of 6.06.5 is maintained by NaOH additions throughout a five day fermentation period at a temperature of 32. The progress of the fermentation is followed by periodic tests for residual sugar and contamination is checked by conventional plating methods.
• Adsorptiom-The crude cobalamin solution contains about 35 mg. LLD active material per liter. Thirtysix liters of this solution are acidified to pH 2.45 with sulfuric acid, filtered, and passed at the rate of ml./min. through 3 columns of Amberlite IRC-SO resin in its hydrogen form, the columns being as described in Example lc and arranged in series. Fifty liters of effluent, including a 14 liter rinse with acidified tap water, contain less than 0.5% of the LLD activity.
• Test I 1000 ml. of the concentrate obtained in step (b) of Example 1 was adjusted to pH 3 with sulfuric acid and to the acidified solution was added 50 g. of Amberlite IRC-50 resin in its hydrogen form. After five hours, the efiluent was filtered from the resin and to the resin was added slowly, with stirring, 2 N sodium carbonate until an amount equal to milliequivalents of sodium carbonate per dry gram of resin had been added. After an agitation period of four hours at room temperature, the mixture was filtered and the resin was washed with several small volumes of water. The eluate and washes were combined and the pH adjusted to 7.5 with sulfuric acid. Water is added to give a final volume of 1000 ml.
• Test II The same procedure as used in Test I was employed, except that no potassium cyanide was added until after completion of the illumination 'period and then 70 mg. of buffered potassium cyanide was added.
• the resulting vitamin B and LLD active components after each key step is shown in the following table.
• Test III 1000 ml. of the concentrate obtained in step (b) of Example 1 was placed, to a depth of one inch, in a white enamel tray. The solution was illuminated for eighteen hours with two standard cool white fluorescent bulbs (15 watts each) placed six inches above the liquid surface. After the illumination period, 70 mg. of buffered potassium cyanide was added and the mixture was then acidified to pH 3 with sulfuric acid and to the acidified solution was added 50 g. of Amberlite IRC-50 resin in its hydrogen form. After six hours, the effluent is filtered from the resin and to the resin is added slowly, with stirring, 2 N sodium carbonate until an amount equal to 10 milliequivalents of sodium carbonate per dry gram of resin had been added.
• Test IV 1000 ml. of the concentrate obtained in step (b) of Example 1 was placed, to a depth of one inch, in a white enamel tray. 35 mg. of buffered potassium cyanide was added and the solution was illuminated for eighteen hours with two standard cool white fluorsecent bulbs (15 watts each) placed 6 inches above the liquid surface. Following illumination, an additional 35 mg. of buffered potassium cyanide was added. The resulting solution was then acidified to pH 3 with sulfuric acid and treated with Amberlite IRC-SO resin as described in Test III. The resulting vitamin B and LLD active components after each key step is shown in the following table.
• a process for preparing vitamin B which comprises culturing a vitamin B producing strain of microorganism in a cobalt containing nutrient medium deficient in cyanide ions, extracting the LLD active components from the medium, treating the extract with a cation exchange resin in its hydrogen form, whereby the LLD active components are adsorbed on the exchanger, eluting said components from the exchanger until the eluate is at a basic pH, and treating the eluate simultaneously for a period of from about 2 to about 30 hours with a source of cyanide ions and light having a wavelength of from about 3600 A. to about 8000 A. to convert the LLD active components to vitamin B 2.
• the exchanger is a methacrylic carboxylic acid cation exchange resin.
• a process for preparing vitamin B which comprises culturing Propionibaczerium freudenreichii in a cobalt containing nutrient medium deficient in cyanide ions, separating the LLD, active components from the resulting Propionibacterium jreudenreichii cells into an aqueous medium, treating said aqueous medium with a cation exchanger resin in its hydrogen form to adsorb the LLD active components thereon, eluting said components from said resin until the eluate is at a basic pH, and treating the eluate simultaneously for a period of from about 2 to about 30 hours with an inorganic cyanide salt and light having a wavelength of from about 3600 A. to about 8000 A. to convert said components to vitamin B 4.
• the resin is a methacrylic carboxylic acid resin.

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Citations (3)

• 1960
  • 1960-08-03 US US47130A patent/US3062723A/en not_active Expired - Lifetime
• 1961
  • 1961-07-31 GB GB27736/61A patent/GB982644A/en not_active Expired
  • 1961-08-03 BR BR131435/61A patent/BR6131435D0/pt unknown

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